Antibiotic combinations of penicillins

ABSTRACT

An antibacterial synergistic composition consisting essentially of a mixture of a penicillin or cephalosporin derivative of the formula ##STR1## and a penicillin of the formula ##STR2## preferably in association with a pharmaceutical carrier.

This is a division of application Ser. No. 464,157, filed Apr. 25, 1974,abandoned which in turn is a continuation-in-part application of Ser.No. 302,423, filed Oct. 31, 1972, now abandoned.

This invention relates to new antibacterial synergistic compositionscontaining penicillin derivatives or cephalosporin derivatives, methodsfor the preparation of such compositions and a method for the treatmentof infectious diseases.

In particular, this invention relates to an antibacterial synergisticcomposition consisting essentially of a mixture of:

(a) a known, clinically useful penicillin or cephalosporin of theformula ##STR3## wherein R is a residue of an organic acid, the residuebeing selected from the group consisting of ##STR4## wherein X isselected from the group consisting of --H, --NH₂, --N₃, --COOH and --SO₃H, and wherein R¹ is selected from the group consisting of the bivalentradicals ##STR5## wherein R₂ is hydrogen, a pharmaceutically acceptablecation or an in vivo rapidly hydrolyzed pharmaceutically acceptableester group, and wherein R₃ is selected from the group consisting ofH--, CH₃ COO--, N₃ --, ##STR6## and

(b) a known, clinically useful penicillin of the formula ##STR7##wherein R₄ is hydrogen, a pharmaceutically acceptable cation or an invivo rapidly hydrolyzed pharmaceutically acceptable ester group, andwherein R₅ and R₆ are lower alkyl groups containing not more than fourcarbon atoms, or R₅ and R₆, when taken together with the adjacentnitrogen atom, represent a ring system of the formula ##STR8## wherein nis 5, 6 or 7.

The above described composition contains the compounds of formula I andII in a weight ratio varying from 10:1 to 1:10, preferably varying from2:1 to 1:2. Optionally, the composition can be incorporated in apharmaceutically acceptable carrier.

It has surprisingly been found that by combining a compound with thegeneral formula I with a compound of the general formula II to form theabove described composition, the antibacterial activity of bothcompounds may be greatly enhanced.

A further surprising finding is that bacterial organisms may developresistance to a combination of compounds of the formula I and II lessreadily than to either of the compounds alone.

In the formula I above the group R is preferably a group of the formula##STR9## wherein X is --H, --NH₂, --N₃, --COOH or --SO₃ H, provided thatthe compound of the formula I is a penicillin. However, when thecompound of the formula I above is a cephalosporin, the group R ispreferably a group selected from ##STR10##

A great number of penicillins and cephalosporins with the generalformula I are known to have strong antibacterial activity and suchpenicillins and cephalosporins have been extensively used for thetreatment of infectious diseases caused by Gram-positive andGram-negative bacteria. In the composition of the invention thepreferred compounds of the formula I are the penicillins:benzyl-penicillin, 6-(D-α-aminophenylacetamido)penicillanic acid,6-(D-α-azidophenylacetamido)penicillanic acid, α-carboxybenzlpenicillin,α-hydroxysulphonyl-benzylpenicillin, and the cephalosporins which areillustrated by the specific combinations of the groups R and R³ in thefollowing table:

    ______________________________________                                        R             R.sub.3         Name                                            ______________________________________                                        NCCH.sub.2    CH.sub.3 COO    Cephacetrile (R.sub.2 = Na)                      ##STR11##                                                                                   ##STR12##      Cephazoline (R.sub.2 = Na)                       ##STR13##    CH.sub.3 COO    Cephalotine (R.sub.2 = Na)                       ##STR14##    H               Cephalexine (R.sub.2 = H)                        ##STR15##                                                                                   ##STR16##      Cephaloridine (R.sub.2 = H)                      ##STR17##    CH.sub.3 COO    EPC 807/2b (R.sub.2  = K)                        ##STR18##    CH.sub.3 COO    EPC 825/1 (R.sub.2 = K)                          ##STR19##    CH.sub.3 COO    EPC 820/1b (R.sub.2 = K)                         ##STR20##    N.sub.3         EPC 821/4II (R.sub.2 = K)                       ______________________________________                                    

as well as the corresponding esters and salts of these penicillins andcephalosporins. In the above table, the designations R, R₂ and R₃ referto the formula I above.

When the composition of the invention is orally administered, thepenicillins or cephalosporins of the formula I may also include suchknown esters theref which are rapidly hydrolyzed in vivo. Examples ofsuch suitable ester groups i.e. the group R₂ in the formula I above, areacyloxy-alkyl groups, e.g. the acetoxy-methyl, the pivaloyloxy-methyl orthe 1"-acetoxy-ethyl group; or alkyloxycarbonyloxy-alkyl groups e.g. theethoxycarbonyloxymethyl or the 1'-ethoxycarbonyloxyethyl group.

The above mentioned suitable ester groups can be described by theformula ##STR21## wherein A is hydrogen or methyl, and B is alkyl oralkoxy. Preferably the group B should not contain more than four carbonatoms. The preferred meaning of the group A and B is methyl and ethoxy,respectively.

Penicillin and cephalosporin esters of this type are known e.g. fromGerman Patent Applications P 21 44 457.5; P 23 12 041.4; P 23 11 328.2;P 23 12 042.5 and P 23 11 346.4.

Penicillins of the general formula II also exhibit strong antibacterialactivity, particularly against Gram-negative organisms. These untilrecently unknown penicillins have been described in Dutch PatentApplication No. 7,016,435 and in British Patent No. 1,293,590.

Examples of compounds of the formula II are6-[(hexahydro-1H-azepin-1-yl)methyleneamino]-penicillanic acid,6-[(piperidyl-1)methylenamino]-penicillanic acid,6-[(hexahydro-1(2H)-azocinnyl)methyleneamino]-penicillanic acid, and6-[(N-ethyl-N-isopropylamino)methyleneamino]-penicillanic acid.

In the composition of the invention the preferred penicillins of theformula II are those wherein the groups R₅ and R₆, when taken togetherwith the adjacent nitrogen atom, represent a ring system of the formula##STR22## wherein n is 5, 6 or 7. Preferably n is 6.

When the composition of the invention is orally administered also thepenicillin of the formula II may be in the form of a known esterthereof, which is rapidly hydrolyzed in vivo. Examples of suitablepenicillin esters included in the formula II are those wherein the groupR₄ is an acyloxy-alkyl group, e.g. the acetoxy-methyl, thepivaloyloxy-methyl or the 1"-acetoxy-ethyl group; or analkyloxycarbonyloxy-alkyl group e.g. the ethoxycarbonylmethyl or the1"-ethoxycarbonyloxyethyl group. Also these ester groups can bedescribed by the formula ##STR23## wherein A and B have the meaninggiven above. Penicillin esters of this type, and included in the formulaII, are known e.g. from Dutch Patent Applications No. 7,016,435 and7,303,434.

Examples of preferred penicillin esters of the formula II arepivaloyloxymethyl 6-[(hexahydro-1H-azepin-1-yl)methyleneamino]penicillanate, acetoxy-methyl6-[(hexahydro-1H-azepin-1-yl)methyleneamino ]penicillanate,pivaloyloxymethyl6-[(hexahydro1(2H)-azocinnyl)methyleneamino]penicillanate,ethoxycarbonyloxymethyl6-[(hexahydro-1H-azepin-1-yl)methyleneamino/penicillanate,1'-ethoxycarbonyloxy-ethyl 6-[(piperidyl-1)methyleneamino]-penicillanate, 1'-acetoxyethyl6-[(hexahydro-1H-azepin-1-yl)-methyleneamino]penicillanate,1'-ethoxycarbonyloxyethyl6-[(hexahydro1(2H)azocinnyl)methyleneamino]penicillanate, and1'-ethoxycarbonyloxy-ethyl6-[(hexahydro-1H-azepin-1-yl)methyleneamino]-penicillanate, andpharmaceutically acceptable salts thereof.

The composition according to the invention can be prepared by variousmixing operations well known for the preparation of compositionscontaining penicillins or cephalosporins. The mixing operations may beaccompanied by chemical reactions between the constituents of the newcomposition. The mixing operations may also be combined with thesimultaneous preparation of esters and salts of the penicillins andcephalosporins included in the composition of the invention. It is alsoto be noted that the new composition according to the invention in somerespects can be regarded as one new chemical individual, as the newcomposition has an antibacterial activity which is unique and widelydifferent from the activity which can be deduced from a calculationbased upon the activity of the single constituents.

The composition of the present invention may be administered eitherorally or by injection. The composition may have optionally incorporatedtherewith other substances, e.g. pharmaceutically acceptable solid orliquid carriers or diluents and may be in any of the conventionalpharmaceutical forms known to the art for pencillin therapy, for examplecompositions suitable for oral administration, for example tablets,granules, capsules, dispersible powders for the preparation of aqueousdispersions for oral use, solutions, suspensions or emulsions, orcompositions suitable for parenteral administration, for example aqueousor nonaqueous solutions or suspensions, or dispersible powders for thepreparation of sterile aqueous dispersions, or compositions suitable fortopical administration, for example ointments.

The compositions according to the invention shown low toxicity and arewell tolerated. In the treatment of bacterial infections in man, thecomposition of the invention is for example administered in amountscorresponding to 5 to 200 mg/kg/day, of the active ingredients of thecomposition, preferably in the range of 10 to 100 mg/kg/day in divideddosages, e.g. two, three or four times a day. They are e.g. administeredin dosage units containing e.g. 175, 350, 500 and 1000 mg of the activeingredients of the composition.

The following examples illustrate the remarkable antibacterialsynergistical effect of the compositions according to the invention.

EXAMPLE 1 In vitro-effect of the combination of6-(D-α-aminophenylacetamido)penicillanic acid and6[(hexahydro-1H-azepin-1yl)methylene amino]penicillanic acid

The in vitro activity of 6-(D-α-aminophenylacetamido)penicillanic acid(I), 6-[(hexahydro-1H-azepin-1-yl)methyleneamino]penicillanic acid (II)and of a combination (III) of equal parts of the two compounds againstclinically isolated enterobacteria was determined in a serial dilutiontest. Tryptose phosphate broth, containing the appropriateconcentrations of the compounds or of the combination of them, wasinoculated with 0.5×10⁴ -5×10⁴ organisms of the various microorganismstested and incubated overnight at 37° C. Minimum inhibitoryconcentrations (M.I.C.) were taken as the concentrations of thecompounds or of the combination at which no visible growth occurred.

    ______________________________________                                                        M.I.C. (μg/ml)                                             Microorganism                                                                             Strain No.                                                                              I         II      III                                   ______________________________________                                        Coliform    3/70      1.56      12.5    0.78                                  Coliform    9/70      3.12      3.12    0.39                                  Coliform    10/70     3.12      3.12    0.78                                  Coliform    17/70     3.12      0.78    0.39                                  Proteus mirabilis                                                                         35895     25        0.78    0.39                                  Proteus vulgaris A    0.78      0.19    0.08                                  Proteus vulgaris B    50        1.56    0.78                                  Proteus     12/70     100       3.12    0.78                                  Proteus     13/70     100       3.12    0.78                                  Proteus     20/70     >100      >100    25                                    ______________________________________                                    

EXAMPLE 2 In vitro-effect of combinations of6-[(hexahydro-1H-azepin-1-yl)-methyleneamino]penicillanic acid withvarious penicillins

Using the same technique as described in Example 1 the in vitro-activityof 6-[(hexahydro-1H-azepin-1-yl) methyleneamino]penicillanic acid (II),of various penicillins and of 1:1-combinations of II with the respectivepenicillin was determined against a clinically isolated coliformbacterium (No. 9/70).

    ______________________________________                                                     M.I.C. (μg/ml)                                                Penicillin     I        II     1:1 combination                                ______________________________________                                        benzylpenicillin                                                                             25       3.12   0.78                                           6-(D-α-azidophenylacet-                                                                50       3.12   1.56                                           amido)penicillanic acid                                                       α-carboxybenzylpenicillin                                                              12.5     3.12   0.38                                           ______________________________________                                    

EXAMPLE 3 In vivo-activity of the combination of 6-(D-α-aminophenylacetamido)penicillanic acid and6-[(hexahydro-1H-azepin-1-yl)methyleneamino]penicillanic acid

Groups of 10 animals of female NMRI white mice, 17-19 g, were givenintraperitoneally inocula of the test bacterium E. coli III andimmediately afterwards treated subcutaneously wth the appropriatediluted aqueous solutions of 6-(D-α-aminophenylacetamido)penicillanicacid (I), 6-[(hexahydro-1H-azepin-1-yl)methyleneamino]penicillanic acid(II) and of a 1:1 combination (III) of the two compounds. The number ofdeaths in the various groups were recorded after 96 hours and the meancurative doses (CD₅₀) were calculated. (In the table, LD₅₀ gives thedilution of an overnight culture of the organism causing a 50% deathrate in animals receiving no therapy; the number of LD₅₀ -doses recordsthe severity of the infections given to the animals.)

    ______________________________________                                                        CD.sub.50 (mg/kg)                                             LD.sub.50                                                                              LD.sub.50 -doses given                                                                     I        II     III                                     ______________________________________                                        1.5 · 10.sup.-3                                                               6.6          5.5      2.0    1.0                                     8.1 · 10.sup.-4                                                               1.2 · 10.sup.4                                                                    250      >500   210                                     ______________________________________                                    

EXAMPLE 4 In vitro-effect of the combination of 6-(D-α-aminophenylacetamido)penicillanic acid and 6[(piperidyl-1)methyleneamino]penicillanic acid

Using the technique described in Example 1, the in vitro activity of6-(D-α-aminophenylacetamido)penicillanic acid (I),6-[(piperidyl-1)-methyleneamino]penicillanic acid (IV) and of acombination (V) of equal parts of the two compounds against clinicallyisolated enterobacteria was determined in serial dilution tests.

    ______________________________________                                                       M.I.C. (μg/ml)                                              Microorganism                                                                            Strain No.                                                                              I         IV      V                                      ______________________________________                                        Coliform   3/70      3.12      3.12    0.78                                              7/70      3.12      1.56    0.78                                              8/70      3.12      6.25    0.78                                              14/70     6.25      3.12    1.56                                              16/70     3.12      1.56    0.78                                              17/70     6.25      1.56    0.78                                   Proteus    4/70      1.56      25      0.78                                              6/70      0.39      3.12    0.18                                              7/70      1.56      50      0.78                                              9/70      6.25      100     1.56                                              10/70     1.56      25      0.78                                   Klebsiella-                                                                              1/70      50        >100    3.12                                   Enterobacter.                                                                            2/70      25        12.5    3.12                                              5/70      100       >100    12.5                                              7/70      >100      >100    100                                               9/70      50        3.12    1.56                                              10/70     50        >100    25                                                431       100       >100    12.5                                   ______________________________________                                    

EXAMPLE 5 In vitro-effect of combination of 6[(piperidyl-1)-methyleneamino]penicillanic acid (IV) with various penicillins

Using the same technique as described in Example 1 the in vitro activityof 6[(piperidyl-1)-methyleneamino]penicillanic acid (IV), of variouspenicillins, and of 1:1 combinations of IV with these penicillins, wasdetermined against a clinically isolated coliform bacterium.

    ______________________________________                                                     M.I.C. (μg/ml)                                                Penicillin     --       IV     1:1 combination                                ______________________________________                                        Benzylpenicillin                                                                             25       6.25   1.56                                           6-(D-α-azidophenylacet-                                                                50       6.25   1.56                                           amido)penicillanic acid                                                       α-Carboxybenzylpenicillin                                                              12.5     6.25   1.56                                           ______________________________________                                    

EXAMPLE 6 Emergence of resistance for E. coli III against6-(D-α-amino-phenylacetamido)penicillanic acid,6-[(piperidyl-1)-methyleneamino]penicillanic acid, and a 1:1 combinationof the two compounds

Subculturing using a serial twofold broth dilution technique wasperformed with 6-(D-α-amino-phenylacetamido)penicillan ic acid (I),6-[(piperidyl-1)methyleneamino]penicillanic acid (IV) and a 1:1combination of I+IV. As inocula were used bacteria from the tube withthe highest concentration of compound still permitting visible growth tothe naked eye after incubation at 37° C. overnight. The followingincreases in MIC-values were noted.

    ______________________________________                                                MIC (μg/ml)                                                        Passage   I             IV     I + IV                                         ______________________________________                                        0         1.56          0.78   0.38                                           1         6.25          6.25   1.56                                           2         25            6.25   0.78                                           3         50            25     0.78                                           4         50            25     1.56                                           5         100           25     3.12                                           6         100           25     3.12                                           ______________________________________                                    

EXAMPLE 7 Emergence of resistance for a coliform microorganism against6-(D)-α-aminophenylacetamido)penicillanic acid,6-[(hexahydro-1H-azepin-1-yl)methyleneamino]penicillanic acid and acombination of the two compounds

Subculturing using a serial twofold broth dilution technique wasperformed with 6-(D-α-aminophenylacetamido)penicillanic acid (I),6-[(hexahydro-1H-azepin-1-yl) methyleneamino]penicillanic acid (II), anda 1:1 combination of I and II. The strain used was a clinically isolatedcoliform. As inocula were used bacteria from the tube with the highestconcentration of compound still permitting visible growth to the nakedeye after incubation at 37° C. overnight. The following increases in MICvalues were noted.

    ______________________________________                                                 MIC (μg/ml)                                                       Passage    I            II     I + II                                         ______________________________________                                        0          1.56         0.39   0.38                                           1          3.12         6.25   0.78                                           2          3.12         25     0.38                                           3          6.25         25     0.78                                           4          6.25         25     0.78                                           5          6.25         25     0.78                                           6          6.25         12.5   1.56                                           7          12.5         25     1.56                                           8          12.5         25     1.56                                           ______________________________________                                    

EXAMPLE 8 In vitro effect of the combination of cephalotin and6-[(N,N-diethylforamidino)-N'-amino]penicillanic acid

Using the technique described in Example 1 the in vitro activity ofcephalotin (VI) and 6-[(N,N-diethylformamidino)-N'-amino]penicillanicacid (VII) and of a combination of equal parts of the compounds againstclinically isolated strains of Coliforma and Klebsiella-Enterobacter wasdetermined in serial dilution tests.

    ______________________________________                                                     Strain                                                                              MIC (μg/ml)                                             Microorganism  No.     VI     VII    VI + VII                                 ______________________________________                                        Coliforma      3       6.25   6.25   1.56                                                    7       12.5   6.25   1.56                                                     14     12.5   12.5   6.25                                                     16     6.25   6.25   3.12                                     Klebsiclla-Enterobacter                                                                      5       25     >100   12.5                                     ______________________________________                                    

EXAMPLE 9 In vitro effect of combinations of 6-(D-α-aminophenylacetamido)penicillanic acid and6-[(N,N-diethylformamidino)-N'-amino]penicillanic acid

Using the technique described in Example 1 the in vitro effect ofcombinations of 6-(D-α-aminophenylacetamido)peni cillanic acid (I) and6-[(N,N-diethylformamidino)-N'-amino]penicillanic acid (VII) in theratios 1:1, 1:2 and 2:1 against clinically isolated strains ofColiforma, Klebsiella-Enterobacter and Proteus was determined in serialdilution tests.

    __________________________________________________________________________                         MIC (μg/ml)                                           Micro- Strain        I + VII                                                                            I + VII                                                                              I + VII                                      organism                                                                             No.  I   VII  1:1  1:2    2:1                                          __________________________________________________________________________    Coliforma                                                                            1    1.56                                                                              3.12 1.56 0.39 + 0.78                                                                          0.78 + 0.39                                         4    3.12                                                                              3.12 3.12 0.78 + 1.56                                                                          1.56 + 0.78                                         7    1.56                                                                              6.25 3.12 0.78 + 1.56                                                                          0.78 + 0.39                                         13   3.12                                                                              12.5 3.12 0.78 + 1.56                                                                          0.78 + 0.39                                         16   3.12                                                                              6.25 1.56 0.78 + 1.56                                                                          0.78 + 0.39                                  Klebsiella-                                                                   Enterobacter                                                                         1    50  >100 12.5 6.25 + 12.5                                                                          12.5 + 6.25                                         5    100 >100 12.5 6.25 + 12.5                                                                          12.5 + 6.25                                         7    >100                                                                              >100 50   25 + 50                                                                              100 + 50                                            8    25  12.5 25   1.56 + 3.12                                                                          6.25 + 3.12                                  Proteus                                                                              9    3.12                                                                              100  3.12 3.12 + 6.25                                                                          1.56 + 0.78                                  __________________________________________________________________________

EXAMPLE 10 In vitro effect of combinations of6-[(hexahydro-1H-azepin-1-yl)methyleneamino]penicillanic acid withvarious cephalosporins

Using the same technique as described in Example 1 the in vitro activityof 6-[(hexahydro-1H-azepin-1-yl) methyleneamino]penicillanic acid (II),of various cephalosporins previously mentioned in this specification,and of 1:1 combinations of II with these cephalosporins was determinedagainst a clinically isolated strain of Klebsiella-Enterobacter (strain4/71)

    ______________________________________                                                 M.I.C. (μg/ml)                                                    Cephalosporin                                                                            --          II      1:1 combination                                ______________________________________                                        Cephacetrile                                                                             50          >100    50                                             Cephazoline                                                                              12.5        >100    6.25                                           Cephalotine                                                                              50          >100    6.25                                           Cephalexine                                                                              50          >100    6.25                                           Cephaloridine                                                                            25          >100    3.12                                           EPC 807 2/b                                                                              >100        >100    12.5                                           EPC 825/1  >100        >100    25                                             EPC 820/1b >100        >100    25                                             EPC 821/4II                                                                              100         >100    6.25                                           ______________________________________                                    

EXAMPLE 11 In vitro effect of combinations of6-[(hexahydro-1H-azepin-1-yl)methyleneamino]penicillanic acid withvarious cephalosporins

Using the same technique as described in Example 1 the in vitro activityof 6-[(hexahydro-1H-azepin-1-yl) methyleneamino]penicillanic acid (II),of various cephalosporins and of 1:1 combinations of II with thesecephalosporins was determined against a clinically isolated strain ofProteus (Mirabilis I 190).

    ______________________________________                                                 M.I.C. (μg/ml)                                                    Cephalosporin                                                                            --        II      1:1 combination                                  ______________________________________                                        Cephacetrile                                                                             12.5      >100    3.12                                             Cephazoline                                                                              6.25      >100    0.78                                             Cephalothine                                                                             3.12      >100    0.78                                             Cephalexine                                                                              25         100    1.56                                             Cephaloridine                                                                            6.25       100    0.78                                             EPC 807 2/b                                                                              25        >100    3.12                                             EPC 825/1  25        >100    1.56                                             EPC 820/1b 50        >100    6.25                                             EPC 821/4II                                                                              12.5      >100    1.56                                             ______________________________________                                    

We claim:
 1. A method of treating bacterial infections in a body whichcomprises forming an antibacterially effective 1:1 mixture of: ##STR24##in the body.
 2. A method of treating bacterial infections in a bodywhich comprises forming an antibacterially effective 1:1 mixture of:##STR25## in the body.